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Adipose cells (sebocytes) are described, The invention especially relates
to sebaceous gland cells and to a sebaceous gland cell line with the
property of being continuously grown over many sub-cultures. The
sebocytes are excellently suited for useful applications.

2. The sebocyte according to claim 1, wherein it is derived from a human
sebaceous gland cell.

3. The sebocyte according to claim 2, wherein the sebaceous gland cell is
a facial sebaceous gland cell.

4. The sebocyte according to claim 1, wherein it is present in the form
of a cell line.

5. The sebocyte according to claim 1, wherein it is immortalized by
transfection of DNA.

6. The sebocyte according to claim 1, wherein it expresses a SV-40 large
T antigen.

7. The sebocyte according to claim 1, wherein it exhibits features of a
normal, non-transfected and differentiating sebocyte.

8. The sebocyte according to claim 1, wherein its proliferation is
modifiable by an antigen and/or a retinoid.

9. The sebocyte according to claim 1, wherein it is cloned.

10. Human sebocyte cell line DSM ACC2383.

11. Use of the sebocyte according to claim 1, for diagnostic, therapeutic
or cosmetic preparations.

12. Use of the sebocyte according to claim 1, for the examination of the
physiology or the pathophysiology of human or animal sebaceous gland.

13. Use of the sebocyte according to claim 1 for the examination of the
origin of acne and/or seborrhoe and/or other diseases.

14. Use of the sebocyte according to claim 13, wherein the other diseases
to be examined are dermal diseases in which the sebaceous gland function
is involved or may be involved.

15. Use of the sebocyte according to claim 1, for the testing of
anti-acne and/or anti-seborrhoe compounds or agents.

16. Use of the sebocyte according to claim 1, for the testing of
compounds or agents against diseases.

17. Use of the sebocyte according to claim 16, wherein the diseases are
dermal diseases in which the sebaceous gland function is involved or may
be involved.

18. Use of the sebocyte according to claim 1, for the development of
simple or complex cell culture systems.

19. Use of the sebocyte according to claim 1, for the formation of, or
for the use in, three-dimensional cell aggregations or constructions of
organ-type structures.

20. Use of the sebocyte according to claim 1, for the preparation of
products derived from said cells.

21. Use according to claim 20, wherein the cell products are lipids,
plasmids, vectors, proteins which are expressed by said cells and/or DNA
or RNA sequences of said proteins.

22. Use of the products obtained according to claim 20 for the
modification of other cells or the modification of organisms.

23. Use of the human sebocyte cell line according to claim 10, for
diagnostic, therapeutic or cosmetic preparations.

24. Use of the human sebocyte cell line according to claim 10, for the
examination of the physiology or the pathophysiology of human or animal
sebaceous gland.

25. Use of the human sebocyte cell line according to claim 10, for the
examination of the origin of acne and/or seborrhoe and/or other diseases.

26. Use of the human sebocyte cell line according to claim 10, for the
testing of anti-acne and/or anti-seborrhoe compounds or agents.

27. Use of the human sebocyte cell line according to claim 10, for the
testing of compounds or agents against diseases.

28. Use of the human sebocyte cell line according to claim 10, for the
development of simple or complex cell culture systems.

29. Use of the human sebocyte cell line according to claim 10, for the
formation of, or for the use in, three-dimensional cell aggregations or
constructions of organ-type structures.

30. Use of the human sebocyte cell line according to claim 10, for the
preparation of products derived from said cells.

31. Use of the products obtained according to claim 21 for the
modification of other cells or the modification of organisms.

Description

STATEMENT OF RELATED APPLICATIONS

[0001] This application is a continuation of U.S. patent application Ser.
No. 14/060,937, filed Oct. 23, 2013, entitled "Sebocytes, Sebocyte-Cell
Line and Uses Thereof", which is a continuation of U.S. patent
application Ser. No. 13/248,370, filed Sep. 29, 2011, entitled
"Sebocytes, Sebocyte-Cell Line and Uses Thereof,", which is a
continuation of U.S. patent application Ser. No. 12/243,869, filed Oct.
1, 2008, entitled "Sebocytes, Sebocyte-Cell Line and Uses Thereof," which
is a continuation of U.S. patent application Ser. No. 09/920,392, filed
Aug. 1, 2001, entitled "Sebocytes, Sebocyte-Cell Line and Uses Thereof,"
all of which are incorporated herein by reference in their entireties.

DESCRIPTION

[0002] The present invention relates to grease or lipid containing and
sebum producing cells of the skin and the mucous membrane, also called
sebocytes. The present invention particularly relates to cells of the
sebaceous gland and a cell line of sebaceous gland with the property of
being capable to be continuously cultured through a large number of
sub-cultures. The sebocytes are particularly suitable for useful
applications, for example for the study of the physiology and the
pathophysiology of human or animal sebaceous glands, for the study of the
generation of acne, seborrhoe or other diseases, for testing the
effectiveness of various substances and of medicaments, for the
development of cell culture systems being based on two-dimensional or
three-dimensional cell assemblies and constructions of organ-like
structures, and for the manufacture of products being derived from these
cells.

BACKGROUND ART

[0003] Increasing indications: suggest that sebocytes play a critical role
in the pathophysiogic processes and diseases of the sebaceous gland/hair
complex, in particular in acne (Gollnick et al. J. Dermatol. 1991;
18:489-499; Brown and Shalita, Lancet 1998;351:1871-1876; Cunliffe,
Dermatology 1998, 196:9-15; Strauss, Dermatology 1998; 126:182-184) The
majority of our understanding of the physiology and pathophysiology of
the sebaceous gland derive from experimental animal models (Pochi in
"Models in Dermatology", Vol 2, N. Lowe and H Maibach, editors, Basel,
1985; 70-75). However, it was found that animal models do not allow
reasonable predictions for the evaluation of the effectiveness of
anti-acne medicaments for humans (Geiger, Dermatology 1995;
1991:305-310). The fact that acne occurs only in humans and that the
secretion activity of the sebaceous gland is strongly species specific
(Nikkari, J Invest. Dermatol. 1974; 257-267) led to the search for human
models. Preliminary studies for the avoidance of these disadvantages was
carried out with human skin samples, which had been either incubated
in-vitro (Hsia et al., Proc. Soc. Exp. Biol. Med. 1970; 135:285-291;
Cooper et al., Br. J. Dermatol. 1976; 94:156-172; Sharp et al., J.
Endrocrinol. 1976; 70:491-499), or had been transplanted to nude mice
(Petersen et al., J Clin. Invest. 1984; 74:1358-1365). Basic studies on
the activity of human sebocytes and their regulation were made possible
only in the recent decade, as vital human sebaceous glands were isolated
(Kealex et al. Br. J. Dermatol. 1986; 114:181-188) and a culture model
for human sebocytes could be established in vitro (Xia et al., J. Invest,
Dermatol. 1989; 93:315-321).

[0005] In spite of these technical improvements, further progress is
strongly hampered by the situation that a cultivation of a large number
of sebocytes from isolated human sebaceous glands is difficult. In
particular, there is the difficulty to keep the cell material in culture
for a long period of time. As the reason therefore, it is assumed that
the sebocytes tend to differentiate and to die via spontaneous cell
membrane rupture and the subsequent release of their content. The best
result yet achieved was that of Fujie et al. (Arch. Dermatol. Res. 1996;
288:703-708), who isolated sebaceous glands on the basis of the technique
of Xia et al. (1989) and cultivated sebocytes by means of the method of
dispersed cell culture through six sub cultures in serum-free,
keratinocyte growth medium without a cell adherence layer.

SUMMARY OF THE INVENTION

[0006] It is an object of the present invention to provide sebocytes
(sebaceous cells) which can be maintained in culture through higher
number of subcultures. In this context, the provided sebocytes shall
approach, in the appearance of their morphological, phenotypic and
functional characteristics, those of viable, normal human sebocytes, at
least to such an extent that they are suitable as cellular material or a
cell culture model for lipid containing, sebum-producing cells, and in
particular for sebocytes, for physiological, pathophysiological and
pharmaceutical evaluations and studies.

[0007] The object is solved by the provision of sebocytes which are
immortalised. Suitably, the cells of the present invention derive from
humans, because this is of primary interest for useful applications.
Sebocytes of this kind are present in the human sebaceous gland cell line
SZ95, which have been deposited with the German collection of
microorganisms and cell cultures (DSMZ) under the depository No. DSM
ACC2383.

DESCRIPTION OF THE DRAWINGS

[0008] FIGS. 1a-1c shows (a) normal, human sebocytes of the second
subculture, from which the provided immortalised sebocytes derive, (b) an
adherent sebocyte culture as provided by immortalised secocytes from the
primary sub culture, and (c) immortalised sebocytes (50th subculture of a
clone). All cells exhibit a similar ephithelial, polymorphous structure.

[0009] FIGS. 2a-c shows cyto-centrifuged samples of (a) immortalised
sebocytes monoclonal antibody against SV40 large T antigene which
resulted in a strong, mostly nucleic, partly cytoplasmatic staining, (b)
Normal keratinocytes and fibroplasts were uniformly negative vis-a-vis
the SV-40 large T protein and the HMEC-1 cells as a positive control
showed mostly a nucleic, partly cytoplasmatic staining for the SV-40
large T protein, while FIG. 2c shows the expression of human SV-40 large
T-antigen in the provided immortalised sebocytes is demonstrated by means
of Western blot analysis.

[0010] FIGS. 3a-b shows the immunocytochemical results on
cytocentrifugation preparations of: (a) the immortalized sebocytes which
exhibited a positive cytoplasmatic staining and (b) normal human
epidermal keratinocytes which were not stained. The preparations were
stained with a monoclonal antibody against the sebaceous gland antigene.

[0011] FIG. 4 shows the expression of the keratines 7, 13 and 19 as well
as various proteins of the human polymorphous epithelial mucine group in
the immortalized sebocytes. Expression was detected by Western blot
analysis, whereas human keratinocytes expressed only keratine 13.

[0012] FIG. 5 shows the staining with Nile Red and the assessment by
fluorescence microscopy showed the presence of lipids in the cell
cytoplasm resulted in individual or in grouped lipid droplets which were
optionally divided in the cytoplasm of the sebocytes.

[0013] FIG. 6 shows HPTLC-fractionized lipids were detected after pulse
recording of radioactively labeled sodium acetate by means of radiometric
image evaluation with two selected immortalized and cloned sebocyte
cultures (see lanes 3 and 4 as well as 5 and 6, respectively).

[0014] FIG. 7 shows the proliferation of an immortalized cloned sebocyte
cell line (SZ95) over 18 days in sebocyte medium.

[0015] FIG. 8 shows an exemplary sebocyte clone where the proliferation of
the cells (seeding of 2.000/well) was observed over 18 days in serum-free
medium (control) as well as in serum-free medium which was supplemented
with 10-6 M 5.alpha.-DHT.

[0017] The present invention will, in the following, be described in more
detail with reference to the drawings (Figs.). FIG. 1 and FIG. 2 show
that immortalised sebocytes SZ95 provided by the invention have
maintained the epithelial, polymorphous appearance of primary, normal
sebocytes, from which they derive (in the present case: from humans). In
addition, the provided immortalised sebocytes and the clones thereof (a
clone means cells which surely derive from a single cell) express the
characteristic 94-kD-large SV-40 large T-antigen, with which coding DNA
sequence transfection had been carried out, even in the later
subcultures. FIG. 1 shows (a) normal, human sebocytes of the second
subculture, from which the provided immortalised sebocytes derive, (b) an
adherent sebocyte culture as provided by immortalised secocytes from the
primary sub culture, and (c) provided immortalised sebocytes (50.sup.th
subculture of a clone) All cells exhibit a similar ephithelial,
polymorphous structure. FIG. 2 shows cyto-centrifuged samples of (a)
provided immortalised sebocytes, and (b) of endothelial cell culture
cells HMEC-1 which served as a positive control, both having been labeled
with a monoclonal antibody against human SV-40 large T-antigen. Both
samples are labeled positively and show that the human SV-40 large
T-antigen is present predominantly in the cell nucleus, partly also in
the cytoplasm of the cell. In (c), the expression of human SV-40 large
T-antigen in the provided immortalised sebocytes is demonstrated by means
of Western blot analysis. While human SV-40 large T-antigen was not
detectable in the non-transfected, normal human sebocytes (lane 1) and
-in normal human ephitelial keratinocytes (lane 2), the characteristic
94-kD large protein was determined in protein extracts of the provided
immortalised sebocytes in the 34.sup.th subculture (lane 3) as well as in
three isolated clones (lanes 4, 5 and 6).

[0018] The immortalised sebocytes of the present invention are preferably
of human origin. The meaning of the expression "sebocytes" is to be
understood in the broadest sense, i.e. relating to all cells which are,
more or less, grease- or lipid-containing and sebum-producing. Sebum is
merely composed of various fatty or lipid substances. In this connection,
the fat or lipid content of the cells may vary in terms of the lipid
substance fraction as well as in terms of content of the lipid substance
fractions. As a rule, but not necessarily, the fatty or lipid substance
content of the cells comprise free fatty acids, triglycerides, wax,
squalene, free cholesterol, cholesterol esters, dihydroxy cholsterol and
other steroids as well as hydrocarbons. In particular, those immortalised
sebocytes are preferred which derive from human sebaceous gland cells. A
particularly good suitability for medicinal purposes is achieved, if the
sebocytes derive from human sebaceous gland cells of the face.

[0019] An essential characteristic of the sebocytes of the invention is
their immortalization Immortalised within the meaning of the present
invention basically means maintaining the vital condition of the cells
through multiple subcultures. The immortalised sebocytes SZ95 of the
present invention could be maintained in culture, in the past observation
time of about 41/2 years, over more than 50 subcultures, whereas normal
human sebocytes can only grow up to three to six subcultures before they
die.

[0020] The immortalised sebocytes according to the present invention can
be obtained by means of transfecting normal sebocytes-using, in the
preferred embodiment, those of human origin and particularly of the human
sebaceous glands--with a DNA which act on forming stable, inactive
complexes with proliferation inhibiting genes. A particularly successful
immortalisation was achieved in the present invention by transfecting
normal human sebocytes, in particular those derived from sebaceous glands
of the face, with a DNA comprising DNA sequences which encode for the
large T-protein of SV-40. The immortalising effect of the SV-40 large
T-antigen (protein) as well as the corresponding use of the coding DNA
sequence for transfecting human cells is basically known. Thus,
immortalised cell lines had been obtained through transfection with a DNA
encoding for SV-40 T, for example with other cells of epithelial origin
(see Tohyama et al., Tohoka. J. Exp. Med. 1997; 182:75-82; Bae et al
Prostate 1998; 34:275-282), as well as of endothelial origin (see Ades et
al., J. Invest. Dermatol. 1992; 99:683-690 and WO-A-92/17569).

[0021] It was found that by transfecting sebocytes with a gene transfer
method by means of applying cationic lipids (LTPOTECTIN reagent, which is
an 1:1 (w/w) liposomal formulation containing the cationic lipids DOTMA
[1,2-Diolyloxy propyl-3-trimethyl ammonium chlorid] and DOPE
[Diolylphosphatidylentanolamin] in membrane filtered water [1 mg/ml]), in
which foreign DNA is taken up through endocytosis into the cells via
cationic lipid/DNA complexes (see Wang et al., In. Vitro. Cell. Dev.
Diol. 1991; 27A:63-74; Staedel et ale, J. Invest. Dermatol. 1994;
102:768-772), good results were achieved for the immortalisation, while
the transfection mixture preferably contained in addition 0.25 to 2.0
vol. -% and particularly 2.0 vol. -% LIPOFECTIN reagent as well as 0.05
to 0.5 wt. -% and particularly 0.5 wt. -% foreign DNA in a suitable
transfection buffer The foreign DNA, like the one coding for the SV-40
large T-protein, is typically inserted in a suitable vector, by which the
expression of the SV-40 large-T-protein is enhanced, preferably by means
of promotor and enhancer sequences. If normal human sebocytes are
transfected, in the preferred embodiment, by the DNA which encodes for
the SV-40 large T-antigen, it is to be expected that the provided
sebocytes express the large T-antigen of SV-40 after a successful
transfection and immortalisation. This was confirmed for the immortalised
sebocytes provided according to the present invention by
immuno-cytochemical means and by means of Western blot analysis, using
monoclonal antibodies against the SV-40 large T-antigen.

[0022] The thus obtained, immortalised sebocytes are preferably in the
state of a cell line which, in this form, can be excellently used for the
application purposes.

[0023] The immortalised sebocytes according to the present invention did
grow, after their adaptation to serum-free culture medium, better than
non-transfected, normal human sebocytes, and they maintained the
capability of synthesising sebaceous gland specific lipids--contrary to
the non-transfected normal human sebocytes which had been maintained in
serum-free medium The immortalised sebocytes according to the present
invention can thus serve as a continuously renewable and propagating cell
line and can grow in defined culture media.

[0024] A particular value of the irmortalised sebocytes according to the
present invention is that they have features of non-transfected, normal
and differentiated sebocytes in morphological, phenotypic and functional
respects Therefore, the immortalized sebocytes according to the present
invention can be excellently offered as models for physiological,
pathophysiological and pharmacological studies At the same time, the
disadvantage of limited viability of conventional, cultured normal
sebocytes of human origin is avoided. Accordingly, it was confirmed that
the immortalised sebocytes according to the present invention can
substantially maintain the phenotype of normal sebocytes and can behave
like non-transfected normal human sebocytes of the face in functional
respects.

[0025] It was found that the immortalised sebocytes or the sebocyte cell
line according to the present invention exhibit a polymorphous,
epithelial appearance which is similar to that of non-transfected, normal
human sebocytes. In cell culture, cells did grow in various sizes and
intracellular structures, which is indicative for various phases in cell
maturation. Thus, cells of various sizes, in the average up to the 5-fold
or 6-fold size with confluent growth, had been observed, which
essentially corresponds to the cell growth increase of non-transfected,
normal human sebocytes with progressive differentiation in vitro (in the
average 4-fold to 5.5-fold size difference). Furthermore, the
immortalised sebocytes according to the present invention was found to be
rich in fatty substance or lipid particles in the cytoplasm, like in
non-transfected, normal human sebocytes. The synthesis of the
characteristic sebaceous gland lipids squalene and wax esters, which are
common for normal human sebocytes, was confirmed experimentally in the
course of the present invention. Furthermore, the immortalized sebocytes
of the present invention synthesised free fatty acids, which, again,
correlates with the findings of non-transfected, normal human sebocytes
in vitro--and even after a high number of subcultures.

[0026] Also, expression markers which confirm a sebocyte origin and which
indicate a viable differentiation, were confirmed as typical indications
for sebocytes of the immortalised sebocytes or sebocyte cell line of the
present invention. Thus, the immortalised sebocytes or the sebocyte cell
line expressed antigens which are typical for the human polymorphous
epithelial mucous protein group, such as the sebaceous gland antigen, the
human milk fat globulins 1 and 2, the human epithelial sialomucine, the
Thomsen-Friedenreich antigen, the mucin-like carcinogen associated
antigen and the epithelial membrane antigen. This was confirmed in the
course of the present invention by immuno cytochemical means and by means
of Western blot analysis. In addition, the immortalised sebocytes or the
sebocyte cell line of the present invention expressed keratinic antigens
typical for non-transfected, normal human sebocytes, such as those of
subclasses 7, 13 and 19. The antigen phenotype thus demonstrated the
sebocyte origin as well as the differentiation of the sebocytes.

[0027] Also in functional terms, the immortalised sebocytes or the
sebocyte cell line of the present invention are similar to
non-transfected, normal human sebocytes. Thus, the immortalised sebocytes
of the present invention responded to the effects of androgens, such as
e.g. by 5.alpha.-dihydro testoterone, by enhancing their in vitro
proliferation. In addition, the immortalised sebocytes or the sebocyte
cell line of the invention possessed the capability of varying their
proliferation by the effects of retinoids, in particular those of the
non-aromatic type (e.g. 13-cis-retinoic acid, all-trans-retinoic acid).

[0028] A preferred embodiment of the present invention is that the
immortalised and preferably the human sebocytes are cloned. This is
advantageous because the immortalised sebocytes or the thus generated
sebocyte cell line are well defined and specifically characterized by
means of their unique genomic basis. A cloned and immortalised human
sebocyte cell line was suitably obtained by gradually diluting
immortalised sebocytes in culture vessels long enough, until the cell
division started again from only one cell per culture vessel. This could
be observed and controlled by means of microscopic observations.

[0029] Accordingly, it was found in the course of the present invention
that the obtained immortalised human sebocytes or the thus obtained
sebocyte cell line essentially maintained the sebocyte identity compared
to non-transfected, normal human sebocytes. This was confirmed by
characteristic determinations and functional tests.

[0030] A sebocyte cell line with the specification SZ95, which entails the
above-mentioned advantages of the present invention, is represented by
the sebaceous gland cell line which was deposited with the DSMZ under the
depository No. ACC2383.

[0031] Accordingly, the immortalised sebocytes or the sebocyte cell line
according to the present invention offer excellent possibilities for
useful applications. In general, the sebocytes or the sebocyte cell line
according to the invention can be used for diagnostic, for therapeutic,
or for cosmetic: uses. Specifically, the sebocytes or the sebocyte cell
line described above may serve for developments and studies of the
physiology or the pathophysiology of lipid-containing, sebum producing
cells, in particular of human or animal sebaceous gland cells, as well as
their role in pathophysiologic processes of the skin and in skin diseases
like, e.g, acne. With the help of the invention the generation of acne
and/or seborrhoe and/or other diseases, especially of skin diseases in
which the sebaceous gland function plays a role or may play a roll, can
be studied The products of the present invention further serve as
excellent models for testing and for evaluating anti-acne compounds
and/or anti-seborrhoe compounds or therapeutic agents, but also
therapeutic agents against diseases, especially skin diseases, in which
the sebaceous gland function plays a role or may play a role. Especially
for the performance of clinical studies, such in vitro studies on
pharmacological properties of medicaments are useful.

[0033] A particularly useful application relates to the generation of
three-dimensional cell assemblies, or constructions or reconstructions of
organ-like structures based on the sebocytes or the sebocyte cell line of
the invention. For this purpose, the sebocytes are used alone, but
preferably in addition to further skin generating cells, in particular
with keratinocytes, fibroblasts, melanocytes, endothelial cells,
Langerhans' cells and/or cells from the hair follicle. For the generation
of three-dimensional cell assemblies, or constructions or reconstructions
of organ-like structures, a support scaffold with collagen or other jelly
materials and/or with parts of inactivated tissue is provided first, and
then the aforementioned cells are applied in or onto this support
scaffold This method is basically known to the man skilled in the art,
and samples are commercially available (Trent and Kirsner, Int. J. Clin.
Pract. 1998; 52:408-413; Fransson et al., Br. T. Dermatol. 1998;
139:589-604; Konstantinova et al., Arch. Dermatol. Res. 1998;
290:610-614; Black et al., FASEB. J. 1998; 12:1331-1340; Zhao et al.,
Biochem. Biophys. Res. Commun 1999; 254:49-53). An "artificial skin" or a
skin substitute is produced thereby, which offers excellent possibilities
for the transplant or grafting medicine, for the reconstruction of
damaged skin portions such as, e.g., burnt skin, or for the therapy of
skin lesions. With the help of the present invention, such an "artificial
skin" can synthesise lipids/sebum in sufficient amounts, when the
sebocytes of the present invention are incorporated into the
constructions.

[0034] A further field of useful applications relates to the manufacture
of products which derive from the sebocytes or the sebocyte cell line of
the invention This includes--the isolation and purification of cellular
substances, such as lipids, proteins, DNA and/or RNA. Since the cells are
immortalised, they are maintained to be offered as a continuous source
for such cellular substances. Specific examples for very suitable
substances, which can be obtained accordingly from these cells, include:
skin lipids for their use in topical agents and medicaments, the
antigenic proteins which are mentioned in Example 3 below in connection
with a phenotypics characterisation of sebocytes, and, further, the
generation of plasmid DNA or vector DNA. The generation of plasmid DNA or
vector DNA is carried out by means of genetic engineering known to those
skilled in the art. Accordingly, genes can be retrieved which induce
lipid production. Especially with such suitable plasmid and vector
constructions, which also includes the generation of viral vectors, again
other cells or organisms can be modified or transfected.

[0035] The present invention will be explained in more detailed by
reference to the following, non-limiting examples.

EXAMPLES

[0036] For the examples described below, the following embodiments as to
the materials and methods may be used--The examples should not be
interpreted as being limiting.

Cell Cultures

[0037] If not indicated otherwise, all cells were maintained in a medium
as adherent cultures which consisted of a modified DMEM/HAM's F12-Medium
(1:1) (available from Biochrom, Berlin, Germany) having 2 mM
N-Acetyl-L-alanyl-L-glutamine which was supplemented with 10%
heat-inactivated, fetal calf serium (FCS; Biochrom) as well as 50
.mu.m/ml gentamycin (available from Gibco-BRL, Karlsruhe, Germany). The
culture was maintained in a humid atmosphere containing 5% CO.sub.2 at
37.degree. C. and the culture medium was renewed every 2 to 3 days.

[0039] Dispersed cells of sub-confluent normal sebocyte cultures were
attached to glass slides by cytocentrifugation. The samples were air
dried and fixed with cold acetone for 10 minutes. The preparations were
subsequently incubated with the respective monoclonal antibody or a
control antibody at room temperature for 30 minutes Bound antibodies were
detected by coupling with a 1:100 dilution of a monoclonal antibody
conjugate from rabbit/anti-mouse Ige (H+L) and an alkaline
phosphatase/anti-alkaline phosphatase complex (available from Dianova,
Hamburg, Germany) at room temperature for 30 minutes. Primary and
secondary monoclonal antibodies were diluted in solutions containing 10%
RPMI-1640 and 10% FCS at a pH of 7.4. The washing steps were conducted
three times with PBS buffer without Ca.sup.2+ and Mg.sup.2+ (available
from Biochrom). The preparations were stained for 30 minutes in buffered
solution (pH 8.8) with Neufuchsin as an adherent agent and a naphthol
salt as a coupling agent (both from Sigma), counter-stained with Mayer's
Haemalum (Merck, Darmstadt, Germany), covered and judged with a light
microscopy.

Isolation and Quantitation of Proteins

[0040] Cell cultures were washed twice with PBS, lysed directly in the
culture dishes by a cold solution which consisted of 50 mM HEPES, 1%
Nonidet P-40 (available from ICN, Aurora, Ohio, USA), 150 mM NaCl as well
as a protease inhibitor (Complete..TM.. Mini; available from Boehringer
Mannheim) subsequently scrubbed and harvested in small centrifugation
dishes to isolate cellular proteins. The obtained material was
homogenized by ultrasonic disruption, subjected to centrifugation and the
supernatants were held on ice, Bicinchoninic acid (BCA-protein assay;
available from Pierce, Rochford, Ill., USA) was added to visualize the
total protein and the protein concentration was quantitated by
measurement of absorption at 550 nm.

Western blot analysis

[0041] Aliquots of the isolated proteins (20 .mu.g) were heated to
95.degree. C. for 15 minutes, One-dimensional SDS/PACE electrophoresis
was conducted with each sample on 7.5% gels. Then, proteins were
transferred to a transfer membrane (Immobilon-P of PVDF; available from
Millipore, Eschborn, Germany) utilizing a standard blot system (available
from Bio-Rad, Munchen, Germany). The blots were incubated with primary
monoclonal antibody at room temperature for 60 minutes and subsequently
with horse radish peroxidase-conjugated goat/anti-mouse monoclonal
antibody and goat/anti-rabbit monoclonal antibody, respectively
(available from Oncogene Science) in a dilution of 0.2 .mu.g/ml at room
temperature for 60 minutes. After thorough washing, the signals were
visualized by a chemiluminescence method utilizing a standard assay (ECL,
available from Amersham, Braunschweig) on X-ray sensitive films (XAR 5;
available from Kodak, Rochester, N.Y., USA), whereby various illumination
intervals were adjusted.

Oil Red and Nile Red Stamina

[0042] Cells grown in chamber slides were incubated either with 0.6% Oil
Red (Sigma) in 60% isopropanol for 15 to 120 minutes or with 1 .mu.g/ml
Nile Red dye (available from Kodak) for 15 minutes at room temperature,
as reported by Xia et al. (1989) (supra). The cultures were then observed
under a light microscope (after Oil Red stain) or a fluorescence
microscope using a 450 to 500 nm bandpass excitation filer by light
emission of >580 nm (after Nile Red stain).

Flow Cytometry

[0043] Dispersed, non-labeled cells were determined for their cell size
using a conventional sorter, while cells labeled with Nile Red dye
(available from Kodak) were assessed for lipid content by flow cytometry
on a fluorescence basis. 10.000 cells per sample were tested.

Labeling and Extraction of Lipids

[0044] _Cell cultures were maintained in culture medium for 2 days and
then radioactively pulsed via the sodium salt of [2.sup.-14 C] acetic
acid (45-60 mCi/mmol; available from DuPont-NEN, Boston, Mass., USA) with
a concentration of 0.5 .mu.ci/ml in RPMI-1640 medium supplemented with 2
mM L-glutamine, 10% heat-inactivated FCS and 100 IU/ml penicilline and
100 .mu.g/ml streptomycine. Incubation was continued for further 24
hours. Lipids were isolated from cultured cells and from the supernatant
culture medium and separated into neutral lipids, fatty acids and
phospholipids (see Seifert et al. J. Invest. Dermatol. 1997; 108: 375).

[0045] The size separation into fractions and the visualization of neutral
lipids and free fatty acids was obtained by high performance thin layer
chromatography (HPTLC) conducted on 20.times.10 cm.sup.2 silica
gel-coated glass plates (available from Merck, Darmstadt, Germany). The
plates were pretreated with n-hexane and dried for 24 hours. The samples
were applied by an automatic lipid applicator (Linomat IV; Camag, Berlin,
Germany). Chromatograms of the neutral lipids were developed in a
n-hexane-diethylether solution (9:1) on 9 cm, dried and post-developed in
a solution of chloroform/diethylether, ethylacetate (80:4:16) on 4-5 cm.
For illumination, illumination sheets (TR 2040S, available from Fuji,
Tokyo, Japan) were used which were scanned using an image analyser ("BAS
1000 Bio-Imaging Analyser", Fuji). Lipid standards were used as
comparative samples.

[0047] To this extent, on the day of evaluation, the culture medium was
removed, the cells were washed twice with PBS and 100 .mu.l of a 100
.mu.l of a 100 .mu.g solution of 4-methylumbelliferyl heptanoate (Sigma,
Deisenhofen, Germany) in PBS were added to each well. The plates were
then incubated at 37.degree. C. for 30 minutes and released fluorescence
was measured by a suitable fluorescence measuring device
(Titertec-Fluoroscan II; Flow, Meckenheim, Germany). Fluorescence units
were obtained at 355 nm excitation and 460 nm emission filters.

[0049] For the treatment with retinoids, all-trans-retinoic acid,
13-cis-retinoic acid and acitretin were dissolved in DMSO and
subsequently placed in serum-free modified DME-Medium/Ham's F12-Medium
(1:1) with 2 nM N-Acetyl-L-alanyl-L-glutamine which was supplemented with
5 ng/ml EGE, 50 .mu.g/ml bovine pituary extract, 1 mg/ml fatty acid-free
bovine serum albumine and 50.mu.g/ml gentamycine to obtain a final
concentration of 10.sup.-7 M retinoid and 0,1% DMSO. 0,1% DMSO alone
served as a control. Retinoids were handled under dim amber light. The
cells (0.5 to 1.times.10.sup.3/well) were treated with the retinoids for
9 days.

Statistical Analysis

[0050] Growth studies were assessed in sextuplicate formulations of 96
well plates. All other experiments were performed in triplicate
formulations.

Example 1

Transfection of Normal Human Sebocytes

[0051] The vector used for the transfection of normal human sebocyte
designated pSVT was a plasmid construct on the basis of PBF322 comprising
sequences for the SV-40 large T protein where its protein expression was
driven by the Rous Sarcoma Virus long-terminal repeat (see Dutt et al.
Oncogene 1990; 5:195-200; Wang et al. In. Vitro. Cell. Dev. Biol. 1991;
27A:63-74). Human sebocyte cultures in the second subculture were grown
to 50% confluency in 35 mm culture dishes (Becton Dickinson, Plymouth,
USA) and used for transfection. The transfection was performed on the
basis of a gene transfer method using cationic lipids. To this extent,
the LIPOFECTIN reagent was utilized which contained a 1:1 (w/w) liposomal
formulation of cationic lipids DOTMA
(1,2-Diolyloxypropyl-3-trimethylammo-niumchloride) and DOPE
(Diolylphosphatidylethanolamine) in membrane filtered water (1 mg/ml). To
this extent, the culture medium was removed, the culture cells were
washed twice with serum-free medium (Opti-MEM from Gibco-BRL) and
incubated in this medium for 4 hours. The medium was then replaced by a
transfection mixture consisting of an antibiotic-free amount of Opti-MEM
medium (1.5 ml) with a suitable amount of the LIPOFECTIN reagent
(Gibco-BRL; 5-30 .mu.l, most preferably 1.5 vol -%) as well as a suitable
amount of pSVT DNA (1-10 .mu.g) in a solution having 0.5 ml PBS (final
DNA concentration, most preferably 0.5 wt. -%). The cultures were
incubated in humid atmosphere containing 5% CO.sub.2 at 37.degree. C. for
24 hours. The cultures were finally washed twice with culture medium and
further maintained in sebocyte culture medium as described above.

[0052] After the transfection treatment, a drastically diminished
viability of the pSVT-treated sebocytes was observed during 4 weeks.
Particularly with the use of optimal amounts of LIPOFECTIN reagent and
pSVT DNA proliferating sebocyte colonies occurred. These cells (SZ95)
were able to be passaged to date more than 50 times. They are still
viable upon the observation period of 4.5 years.

[0053] Example 2

Cloning of Immortalized Human Sebocytes

[0054] The thus immortalized human sebocytes SZ95 were seeded in 96 well
culture plates using a dilution series with geometrically descending cell
numbers of 1.times.10.sup.2 cells in the first series until theoretically
zero cells were reached in the last series (Zouboulis et al. in "The
malignous melanome of the skin", C. E. Orfanos and C. Garbe (eds.)
Zuckschwerdt, Munchen, Germany: 1990; 158-168). The cells were maintained
in standard culture medium supplemented with 5 mg/ml ECF and 3 ng/ml KGF.
Growing cells were regarded as clones in case they were derived from a
single cell per well which was observable by light microscopic
experiments. Thus, cloned SZ95 cells were obtained.

[0055] Example 3

Characterization of the Immortalized Human Sebocytes

Detection of SV-40 Large T antigene

[0056] The expression of SV-40 large T Antigene in immortalized sebocytes
was detected immunocytochemically and by Western blot analysis using a
monoclonal anti-human SV-40 large T antigene antibody from mouse serum
(Oncogene Science, Cambridge, Mass., USA) which was diluted for
immunocytochemical analysis to 1:1000 and for Western blot analysis to
1:100. Human normal epidermal keratinocytes, dermal fibroplasts and as a
positive control endothelial cells HMEC-1 immortalized by SV-40 large T
antigene (see WO-A-992/175 69) were used as a comparison.

[0057] The immunocytochemical experiment of the immortalized sebocytes
according to Example 1 with the monoclonal antibody against SV40 large T
antigene resulted in a strong, mostly nucleic, partly cytoplasmatic
staining (see FIG. 2a). Normal keratinocytes and fibroplasts were
uniformly negative vis-a-vis the SV-40 large T protein and the HMEC-1
cells as a positive control showed mostly a nucleic, partly cytoplasmatic
staining for the SV-40 large T protein (see FIG. 2b).

[0058] FIG. 2c shows the results of Western blot analysis of SV-40 large T
antigene expression in non-transfected normal human sebocytes (lane 1),
in normal human epidermal keratinocytes (lane 2), in immortalized
sebocytes according to the present invention (34th sub-culture; lane 3)
as well as in various cloned sebocytes according to the present invention
(lanes 4, 5 and 6). A band at 94 kD was confirmed to be the immortalized
sebocyte line as well as its clones which confirmed the expression of the
SV-40 large T protein (see Harlow et al. J. Virol. 1981; 39:861-869).

Phenotypic Characterization of the Immortalized Sebocytes According to the
Present Invention

[0059] The morphology of the immortalized sebocytes SZ95 according to
Example 1 was epithelial and exhibited a polymorphous appearance with
cells of different size, whereas numerous droplets could he observed in
the cytoplasm (see FIGS. 1b and 1c).

[0060] Immunocytochemical experiments of the immortalized sebocytes
according to the present invention with respective antibodies resulted in
a positive finding against the sebaceous gland antigene in contrast to
normal epidermal keratinocytes which were not stained by the monoclonal
antibody against the sebaceous gland antigene (see FIG. 3). FIG. 3 shows
the immunocytochemical results on cytocentrifugation preparations (a) of
the immortalized sebocytes according to the present invention as well as
(b) normal human epidermal keratinocytes. The preparations were stained
with an monoclonal antibody against the sebaceous gland antigene While
the immortalized sebocytes of the present invention exhibited a positive
cytoplasmatic staining, the normal human epidermal keratinocytes were not
stained.

[0062] The staining with Nile Red and the assessment by fluorescence
microscopy showed the presence of lipids in the cell cytoplasm. In the
immortalized sebocytes SZ95 of the present invention (see FIG. 5),
stained by Nile Red fluorescent dye directed to neutral lipids resulted
in individual or in grouped lipid droplets which were optionally divided
in the cytoplasm of the sebocytes The immortalized sebocytes according to
the present invention decreased their content from 510 fluorescent units
per cell (median value) in serum-containing medium to 429 fluorescent
units per cell (median value; i.e. --16%) in serum-free medium detected
by fluorescence cytometric experiments of cells stained with Nile Red.

[0063] The immortalized sebocytes according to the present invention of
Example 1 synthesized various fractions of neutral lipids including the
typical sebaceous gland lipids squalene and wax ester as well as
triglycerides, cholesterol, cholesterol ester, diglycerides, lanosterol
and free fatty acids. This was observed throughout 25 to 40 sub-cultures.
The result in shown in FIG. 6, where HPTLC-fractionized lipids were
detected after pulse recording of radioactively labeled sodium acetate by
means of radiometric image evaluation with two selected immortalized and
cloned sebocyte cultures (see lanes 3 and 4 as well as 5 and 6,
respectively). As shown by lanes 3, 4 and 5, the cells synthesized
multiple fractions of neutral lipids including squalene (Sq), wax ester
(WE) as well as triglycerides (Tg), cholesterol (Cho), cholesterol ester
(ChE), diglycerides (Dg), lanosterol (Lan) as well as free fatty acids
(FFA). All neutral lipids were also found but in a lesser extend in the
extracellular supernatant (see lane 6). For comparison in lane 1, lipid
standards, in lane 2 human sebum, and in lane 4, free fatty acids
extracted from cells were applied. As further comparison, lanes 7 and 8
showed the results of keratinocytes, whereas lane 7 showed the presence
mainly of cholesterol and triglycerides in the cells, while from the
supernatant (lane 8), mostly cholesterine was found.

Proliferation Studies

[0064] _A logarithmic proliferation pattern of the immortalized sebocytes
according to the present invention was detected under normal culture
conditions with population doubling times of 52.4+-0.1.6 independent from
the original culture cell densities. To this extent, FIG. 7 shows the
proliferation of an immortalized cloned sebocyte cell line (SZ95) over 18
days in sebocyte medium.

[0065] The proliferation of the immortalized sebocytes was reduced after
addition of serum-free medium but was retrieved after addition of
5.alpha.-DHT. This is shown in FIG. 8 for an exemplary sebocyte clone
where the proliferation of the cells (seeding of 2.000/well) was observed
over 18 days in serum-free medium (control) as well as in serum-free
medium which was supplemented with 10.sup.-6 M 5.alpha.-DHT. After the
8.sup.th day, 5.alpha.-DHT increased the proliferation of the cells
significantly which was shown by the determined population doubling time
of 136 hours (control) and 53.7 hours (5.alpha.-DHT-treated cells (*,
p<0.05; **, p<0.01).

[0066] The influence of retinoids on the immortalized cells showed a
differentiated response of the proliferation behavior. While some clones
showed inhibition of proliferation by retinoids (typically distinctively
pronounced in the order of 13-cis-retinoic acid>all-trans-retinoic
acid>>Acitretin), other clones were stimulated in proliferation
(for example by all-trans-retinoic acid and 13-cis-retinoic acid)
corresponding to the proliferation response of normal human epidermal
keratincoytes. This is shown in FIG. 9 (*, p<0.05; **, p<0.01; ***,
p<0.001).